The present invention relates generally to oligonucleotide synthesis and, more particularly, to a new and improved primer system that enables oligonucleotides to be more easily and more efficiently synthesized on a solid support.
Oligonucleotides are relatively short pieces of either DNA (deoxyribonucleotides) or RNA (ribonucleotides) with chain lengths in the range of from 3-100 base units. Both deoxyribonucleotides and ribonucleotides have particular biological significance due to their key roles in cellular processes and cell growth. The component of the cell which contains the primary information for growth and protein expression is DNA. Due to the fact that it is now possible to incorporate newly synthesized pieces of DNA into the DNA of a cell, methods which facilitate the chemical synthesis of oligodeoxyribonucleotides take on particular significance. Such methods can be employed either to help correct defective genetic information or to substantially modify the proteins which an organism expresses. For example, a bacterium may be provided with the polydeoxyribonucleotide which contains the genetic information for the synthesis of human glucagon. Under the proper conditions, the organism would then produce human glucagon.
In addition, a range of other pharmacological, diagnostic and research applications of oligonucleotides exist. The full usefulness of oligonucleotides, however, awaits methods to effectively synthesize them in high yield and greater purity.
Until the mid-1970's, oligonucleotide synthesis was carried out in a liquid phase. The separation and purification problems associated with liquid phase techniques prevented a practical automated system for oligonucleotide synthesis. To solve these separation and purification problems, polymeric supports were developed. Up until now, use of these polymeric supports has involved the coupling of the oligonucleotide to the solid support by procedures which permit cleavage between the support and the first nucleotide. However, since the first nucleotide can be any one of four bases, these procedures have necessitated the implementation of eight different initiated supports, i.e., four for DNA and four for RNA, in order to synthesize desired oligonucleotides. It would be a significant advancement if a support system were available, with a primer (chain initiator) of great versatility, such that all desired oligonucleotides could be synthesized from a single type of polymeric support.
To be of practical utility, a polymeric support and primer must retain the growing oligonucleotide until synthesis is complete. Also, once synthesis is complete, the primer should be capable of being cleaved to permit the release of the oligonucleotide from the polymeric support. Many of the primers discussed in the literature possess linkages that are very acid or base labile. Thus, the oligonucleotide may be released from the polymeric support, at an undesirable time with resulting structural rearrangement, by treatment with acidic or basic reagents. See, e.g., M. D. Matteucci and M. H. Caruthers, Synthesis of Deoxyoligonucleotides on a Polymer Support, Journal of the American Chemical Society, Vol. 103, No. 11, 1981, pgs. 3185-3191, and H. Sommer and F. Cramer, Chemische Synthese von Desoxyoligonucleotides mit 5-Phosphatgruppe am Polymeren Trager (Chemical Synthesis of Deoxyoligonucleotides with 5-Phosphate Group on a Polymer Support), Chem. Ber., Vol. 107, 1974, pgs. 24-33. The use of an acid labile primer, however, prevents the use of mildly acidic reagents or conditions during the oligonucleotide synthesis. Similarly, use of a base labile primer prevents the use of mildly basic reagents or conditions. Consequently, using an acid or base labile primer greatly restricts the versatility of a polymeric support system.
Primers that are very acid or very base labile also restrict the versatility of a polymeric support system in other ways. For instance, many blocking groups are ordinarily used to protect the various functions of the nucleotides, i.e., the amine or hydroxyl functions of the base, the 2' hydroxyl of ribonucleotides, the 3' and the 5' hydroxyls of deoxyribonucleotides and ribonucleotides, and the phosphate groups. Preferably, the polymeric support system is versatile enough to permit removal of these protecting groups before the oligonucleotide is released from the polymeric support or, in the alternative, the oligonucleotide can be released from the polymeric support before the protecting groups are removed. Primers that are very acid labile or very base labile, however, significantly restrict this versatility.
What is needed, therefore, is a completely versatile polymeric support system, that is, a polymeric support arrangement and corresponding method of oligonucleotide synthesis that can withstand mildly basic and mildly acidic reaction conditions and still permit a convenient and quantitative release at the desired time of all types of synthesized oligonucleotides from a single polymeric support. The present invention satisfies this need.